Composition and method for detecting pseudomonas plecoglossicida based on CRISPR / Cas12a

Abstract

The invention relates to the technical field of bacterial detection, and discloses a composition and a method for detecting pseudomonas plecoglossicida based on CRISPR / Cas12a (Clustered Regularly Interspaced Short Palindromic Repeats / Cas12a). The composition comprises a primer group, crRNA (Complementary Ribonucleic Acid) and Cas12a protein, and the crRNA comprises a sequence as shown in SEQ ID NO. 3. When the composition disclosed by the invention is used for detecting the pseudomonas plecoglossicida, relatively high sensitivity and specificity can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial detection, and in particular to a composition and method for detecting Pseudomonas aeruginosa based on CRISPR / Cas12a. Background Art

[0002] Pseudomonas plecoglossicida is an important bacterial pathogen that harms the aquaculture industry. It can infect a variety of fish hosts, including freshwater and marine fish. It mainly causes sepsis and ascites in freshwater fish such as Plecoglossus altivelis, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius auratus); it mainly causes visceral white spot disease in marine fish such as large yellow croaker (Larimichthys crocea) and cogtail grouper (Epinepheluscoioides). White nodules and granulomas appear in the liver, kidney, spleen and other organs of the infected fish. After the disease occurs, it will cause large-scale mortality of fish and cause great economic losses to the aquaculture industry. At present, there are no very effective drugs and vaccines for the prevention and control of Pseudomonas plecoglossicida. Therefore, in the context of vigorously advocating ecological, green and efficient development, early quarantine of the disease is particularly critical. Comprehensive pathogen inspection of fish species is an important part of early prevention and control of the disease.

[0003] Pseudomonas aeruginosa belongs to the genus Pseudomonas. Due to the slow evolution of Pseudomonas, the sequence homology of 16S ribosomal RNA of Pseudomonas aeruginosa is high and it is difficult to distinguish between them. Therefore, one of the difficulties in developing a detection method is to distinguish Pseudomonas aeruginosa from other Pseudomonas.

[0004] The traditional bacterial isolation and culture method is the standard method for detecting Pseudomonas aeruginosa, but this method is time-consuming and usually takes 2 to 3 days to complete. At present, some new detection technologies for Pseudomonas aeruginosa have been developed, mainly molecular detection methods based on nucleic acid or antigen-antibody reactions. However, these detection methods require special instruments and professional operators, and the detection sensitivity needs to be improved, making it difficult to achieve accurate detection in the early stages of the disease. Therefore, the development of a rapid, sensitive and visual detection system for Pseudomonas aeruginosa is of great significance for its early warning and prevention and control. Summary of the invention

[0005] In order to solve the technical problem that the existing detection technology of Pseudomonas aeruginosa is difficult to achieve high sensitivity and portability at the same time, the present invention provides a composition and method for detecting Pseudomonas aeruginosa based on CRISPR / Cas12a. The composition of the present invention can achieve high sensitivity, specificity and portability when detecting Pseudomonas aeruginosa.

[0006] The specific technical scheme of the present invention is: In a first aspect, the present invention provides a composition for detecting Pseudomonas aeruginosa based on CRISPR / Cas12a, comprising a primer set, crRNA and Cas12a protein; the crRNA comprises a sequence as shown in SEQ ID NO.3.

[0007] The present invention designs a CRISPR / Cas12a detection system for Pseudomonas aeruginosa. CRISPR / Cas12a detection technology is based on crRNA and Cas12a as the main components, and activates the nonspecific shearing activity of Cas12a through targeted binding of crRNA and dsDNA, and can specifically identify the target gene. The detection method based on the CRISPR / Cas12a system can specifically cut the target gene and quickly develop color after the reaction. Before performing CRISPR-Cas12a detection, the target gene is amplified first, which can improve the detection sensitivity.

[0008] In the CRISPR / Cas12a detection system, the design of crRNA has an important impact on the detection sensitivity and specificity. The present invention uses crRNA with a sequence as shown in SEQ ID NO.3, which can achieve higher detection sensitivity, is conducive to the accurate detection of low-killing Pseudomonas aurantii content samples, and at the same time, has better detection specificity and can avoid interference from multiple non-target bacteria.

[0009] Preferably, the primer set is a PCR primer set, comprising: an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.2.

[0010] Preferably, the primer set is an RPA primer set, comprising one of the following a) to c): a) an upstream primer shown in SEQ ID NO.6 and a downstream primer shown in SEQ ID NO.7; b) an upstream primer shown in SEQ ID NO.8 and a downstream primer shown in SEQ ID NO.9; c) an upstream primer shown in SEQ ID NO.10 and a downstream primer shown in SEQ ID NO.11.

[0011] The use of the above-mentioned specific sequence PCR primer set or RPA primer set for nucleic acid amplification can cooperate well with the specific crRNA in the present invention to achieve high sensitivity and high specificity detection of Pseudomonas aeruginosa.

[0012] Preferably, the composition further comprises a lateral flow chromatography test strip.

[0013] When the composition of the present invention is used to detect Pseudomonas aeruginosa, the detection result can be visualized by observing the fluorescent signal or detecting with a lateral flow chromatography test strip, wherein the latter can reflect the detection result more conveniently and intuitively.

[0014] Preferably, the composition further comprises a reporter molecule; the sequence of the reporter molecule is: 5'-FAM / TTTATTT / 3'-BHQ1 or 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin.

[0015] Preferably, in the lateral flow chromatography test strip: the conjugate pad contains AuNPs-Anti-FAM, the detection line is bound to a secondary antibody, and the quality control line is bound to streptavidin; the composition also includes a reporter molecule with a sequence of 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin.

[0016] In a second aspect, the present invention provides a method for detecting Pseudomonas aeruginosa for non-diagnostic purposes using the composition, comprising the following steps: 1) preparing a nucleic acid amplification system comprising the primer set, and performing nucleic acid amplification using the DNA extracted from the sample to be tested as a template to obtain an amplified product; 2) preparing a CRISPR / Cas12a reaction system comprising the crRNA and Cas12a protein, performing a CRISPR / Cas12a reaction using the amplified product as a template, and judging whether Pseudomonas australis is present in the sample to be tested according to the product.

[0017] Preferably, in step 2), the CRISPR / Cas12a reaction system also includes a reporter molecule with a sequence of 5'-FAM / TTTATTT / 3'-BHQ1; the method for judging whether Pseudomonas aeruginosa exists in the sample to be tested based on the product is as follows: observing the product under ultraviolet light, the product showing green or light green fluorescence indicates that Pseudomonas aeruginosa exists in the sample to be tested, and the product showing colorless fluorescence indicates that Pseudomonas aeruginosa does not exist in the sample to be tested.

[0018] Preferably, in step 2), the CRISPR / Cas12a reaction system also includes a reporter molecule with a sequence of 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin; the method for judging whether Pseudomonas aeruginosa is present in the sample to be tested based on the product is as follows: the product is added to a lateral flow chromatography test strip, and the detection line shows red, indicating that Pseudomonas aeruginosa is present in the sample to be tested, and only the quality control line shows red, indicating that Pseudomonas aeruginosa is not present in the sample to be tested.

[0019] Preferably, in step 1), the nucleic acid amplification system is a PCR amplification system or an RPA amplification system.

[0020] Preferably, in step 2), the CRISPR / Cas12a reaction system further comprises NEBuffer 2.1.

[0021] Compared with the prior art, the present invention has the following advantages: (1) The composition of the present invention can achieve a high detection sensitivity when detecting Pseudomonas aeruginosa, which is conducive to the accurate detection of samples with low Pseudomonas aeruginosa content. According to the test, the detection limit of the composition of the present invention can reach 2.34×10 2 Copy / React.

[0022] (2) The composition of the present invention has good specificity in detecting Pseudomonas aeruginosa. According to experiments, the composition of the present invention can avoid interference from a variety of non-target bacteria including Pseudomonas aeruginosa (P. aeruginosa), Pseudomonas putida (P. putida), Pseudomonas fluorescens (P. fluorescens), Vibrio parahaemolyticus (V. parahaemolyticus), Edwardsiella tarda (E. tarda), Vibrio alginolyticus (V. alginolyticus), Vibrio harveyi (V. harveyi), Streptococcus iniae (S. iniae) and Nocardia seriolae (N. seriolae).

[0023] (3) The composition of the present invention can be used to reflect the test results through a lateral flow chromatography test strip, which takes a short time and is simple and portable to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 For the design and effect comparison of crRNA. Figure 1 A is a flow chart of PCR and CRISPR / Cas12a reactions in Example 1; Figure 1 B is the targeting position of the three crRNAs on the gyrB gene; Figure 1 C is the CRISPR / Cas12a reaction results of three crRNAs. Figure 1 In C, “ssDNA activator” is a single-stranded DNA that is reverse complementary to crRNA.

[0025] Figure 2 The test results of the RPA-CRISPR / Cas12a detection method. Figure 2 A is the agarose gel electrophoresis of the amplification products of three sets of RPA primers; Figure 2 B shows the results of RPA-CRISPR / Cas12a using different RPA primers; Figure 2 C is the sensitivity test result of RPA-CRISPR / Cas12a (the result was observed under ultraviolet light using a gel imager); Figure 2 D is the sensitivity test result of RPA-CRISPR / Cas12a (fluorescence changes were monitored using a real-time fluorescence quantitative PCR system); Figure 2 E is the specific detection result of RPA-CRISPR / Cas12a.

[0026] Figure 3 The test results of the RPA-CRISPR / Cas12a-LFS detection method. Figure 3 A is a flow chart of the RPA-CRISPR / Cas12a-LFS method; Figure 3 B is the sensitivity test result of RPA-CRISPR / Cas12a-LFS; Figure 3 C is the specific detection result of RPA-CRISPR / Cas12a-LFS.

[0027] Figure 4 Results of using RPA-CRISPR / Cas12a-LFS to detect spiked samples and clinical samples. Figure 4 A is the test result of spiked sample (gel imager); Figure 4 B is the test result of spiked sample (real-time fluorescence quantitative PCR system); Figure 4 C is the test result of spiked sample (LFS); Figure 4 D is the test result of clinical samples (gel imager); Figure 4 E is the test result of clinical samples (real-time fluorescence quantitative PCR system); Figure 4 F is the clinical sample test result (LFS). DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the embodiments.

[0029] Overall embodiment A composition for detecting Pseudomonas australis based on CRISPR / Cas12a, comprising a primer set, crRNA and Cas12a protein; the crRNA comprises a sequence as shown in SEQ ID NO. 3. The present invention adopts a specific crRNA, which can achieve higher detection sensitivity, is conducive to the accurate detection of samples with low Pseudomonas australis content, and at the same time, has better detection specificity, can avoid interference from multiple non-target bacteria, and uses the CRISPR / Cas12a detection system to achieve better convenience.

[0030] As a specific embodiment, the primer set is a PCR primer set, including: an upstream primer shown in SEQ ID NO. 1 and a downstream primer shown in SEQ ID NO. 2. The PCR primer set with the above specific sequence is used for nucleic acid amplification, which can cooperate well with the specific crRNA in the present invention to achieve high sensitivity and high specificity detection of Pseudomonas aeruginosa.

[0031] As another specific embodiment, the primer set is an RPA primer set, comprising one of the following a) to c): a) an upstream primer shown in SEQ ID NO.6 and a downstream primer shown in SEQ ID NO.7; b) an upstream primer shown in SEQ ID NO.8 and a downstream primer shown in SEQ ID NO.9; c) an upstream primer shown in SEQ ID NO.10 and a downstream primer shown in SEQ ID NO.11.

[0032] The use of the above-mentioned specific sequence PCR primer set for nucleic acid amplification can cooperate well with the specific crRNA in the present invention to achieve high-sensitivity and high-specificity detection of Pseudomonas aeruginosa.

[0033] As a specific embodiment, the composition also includes a lateral flow chromatography test strip. When the composition of the present invention is used to detect Pseudomonas aeruginosa, the detection result can be visualized by observing the fluorescent signal or by using a lateral flow chromatography test strip, wherein the latter can reflect the detection result more portable and intuitively.

[0034] As a specific embodiment, the composition further includes a reporter molecule; the sequence of the reporter molecule is: 5'-FAM / TTTATTT / 3'-BHQ1 or 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin.

[0035] The test shows that the detection limit can reach 2.34×10 2The system can effectively avoid interference from a variety of Pseudomonas and common pathogens of marine fish, including Pseudomonas aeruginosa (P.aeruginosa), Pseudomonas putida (P.putida), Pseudomonas fluorescens (P.fluorescens), Vibrio parahaemolyticus (V.parahaemolyticus), Edwardsiella tarda (E.tarda), Vibrio alginolyticus (V.alginolyticus), Vibrio harveyi (V.harveyi), Streptococcus iniae (S.iniae) and Nocardia seriolae.

[0036] A method for detecting Pseudomonas aeruginosa for non-diagnostic purposes using the composition comprises the following steps: 1) preparing a nucleic acid amplification system comprising the primer set, performing nucleic acid amplification using DNA extracted from a sample to be tested as a template, and obtaining an amplified product; 2) preparing a CRISPR / Cas12a reaction system comprising the crRNA and Cas12a protein, performing a CRISPR / Cas12a reaction using the amplified product as a template, and judging whether Pseudomonas australis is present in the sample to be tested according to the product.

[0037] As a specific implementation, in step 1), the nucleic acid amplification system is a PCR amplification system or an RPA amplification system.

[0038] As a specific embodiment, in step 2), the CRISPR / Cas12a reaction system also includes NEBuffer2.1.

[0039] As a specific implementation, in step 2), the CRISPR / Cas12a reaction system also includes a reporter molecule with a sequence of 5'-FAM / TTTATTT / 3'-BHQ1; the method for judging whether Pseudomonas aeruginosa is present in the sample to be tested based on the product is as follows: observing the product under ultraviolet light, the product showing green or light green fluorescence indicates that Pseudomonas aeruginosa is present in the sample to be tested, and the product showing colorless fluorescence indicates that Pseudomonas aeruginosa is not present in the sample to be tested.

[0040] As another specific implementation, in step 2), the CRISPR / Cas12a reaction system also includes a reporter molecule with a sequence of 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin; the method for judging whether Pseudomonas aeruginosa exists in the sample to be tested based on the product is as follows: the product is added to a lateral flow chromatography test strip, and the detection line shows red, indicating that Pseudomonas aeruginosa exists in the sample to be tested, and only the quality control line shows red, indicating that Pseudomonas aeruginosa does not exist in the sample to be tested. Specific embodiments The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0042] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0043] Example 1: Comparison of the effects of different crRNAs The experimental process of this embodiment is as follows Figure 1 As shown in A, the specific gene gyrB is amplified first, and the crRNA specifically targeting the gyrB gene is designed. When Cas12a binds to the specific dsDNA (gyrB) guided by crRNA, its nonspecific trans-cleavage activity is activated, cutting the FAM-N7-BHQ1 ssDNA probe, thereby stimulating fluorescence. The details are as follows: 1.1 Amplification of target gene fragments First, extract the genomic DNA of Pseudomonas aeruginosa, and then use it as a template to amplify the target gene fragment by PCR. The amplification system is as follows: PCR upstream primer: 5'-GACAACTCGTACAAGGTTTCTGGCG-3' (SEQ ID NO. 1); PCR downstream primer: 5'-GGAAACCAGCTTGTCCTTGGTCTG-3' (SEQ ID NO. 2).

[0044] PCR reaction system: 0.25μL TaKaRa Ex Taq, 2.5μL 10×Ex Taq Buffer, 2μL dNTPMixture, 1μL PCR upstream and downstream primers, 2μL Pseudomonas aeruginosa genomic DNA, add pure water to make the final volume 25μL; PCR reaction conditions: 95℃ for 5 minutes; 95℃ for 30 seconds, 61℃ for 30 seconds, 72℃ for 40 seconds, for 30 cycles; 72℃ for 10 minutes.

[0045] The PCR product was used as a template for subsequent target gene fragments.

[0046] 1.2 CRISPR / Cas12a reaction Synthesize 3 kinds of crRNAs targeting different positions of gyrB gene (target positions on gyrB gene are as follows Figure 1 B), and the sequence is shown in Table 1. In Table 1, uracil in each RNA sequence is represented by the letter "U" commonly used in the art; in the sequence listing file, according to the ST.26 standard, uracil in each RNA is represented by the letter "T", which has the same meaning as the letter "U" in the sequence in Table 1.

[0047] Table 1 crRNA sequences The target gene fragment obtained by PCR amplification was used as a template for the CRISPR / Cas12a reaction. The CRISPR / Cas12a reaction system is as follows: 100nM LbCas12a, 2μL 10×NEBuffer 2.1, 400nM crRNA, 1000nM FAM-N7-BHQ1ssDNA (single strand DNA) probe, 3μL PCR product, and pure water was added to make the final volume 20μL. Among them, the crRNAs used are crRNA1, crRNA2 and crRNA3 in Table 1; the sequence of the reporter molecule FAM-N7-BHQ1 used is: 5'-FAM / TTTATTT / 3'-BHQ1. The ssDNA activator was used as a positive control. The ssDNA activator is a single-stranded DNA that is reverse complementary to crRNA. After reacting at 37°C for 30 minutes, the reaction results were visualized under ultraviolet light using a gel imager. If the reaction solution shows green or light green fluorescence, the result is judged as positive; if the reaction solution appears colorless, it is judged as negative.

[0048] result( Figure 1 C shows the CRISPR / Cas12a reaction results of three crRNAs) shows that in the presence of DNA template and positive ssDNA activator, under ultraviolet light, the system using crRNA1 and crRNA2 can emit green fluorescence (shown as green in the original figure, because the attached figure cannot have color, so Figure 1 C is shown in gray), while the negative control does not show any fluorescent signal. crRNA1 was randomly selected for subsequent CRISPR / Cas12a detection. In the following, unless otherwise specified, the crRNA used is crRNA1.

[0049] Example 2: Detection sensitivity and specificity of RPA-CRISPR / Cas12a 2.1 Amplification of target gene fragments First, the gyrB gene sequence (accession number: AB178862.1) was connected to the pUC19 plasmid to obtain the gyrB-pUC19 plasmid, which was then used as a template to amplify the target gene fragment by RPA. The amplification system was as follows: Three sets of RPA upstream and downstream primers were used, and their sequences are shown in Table 2 ; RPA reaction system: 2 μL upstream and downstream primers (10 mM), 2 μL gyrB-pUC19 and RPA composition, add pure water to make the final volume 50 μL; RPA reaction conditions: 37°C for 30 minutes.

[0050] Table 2 RPA primer sequences The RPA product was detected by agarose gel electrophoresis. Figure 2 A shows the agarose gel electrophoresis of the products amplified by three sets of RPA primers) showing that the DNA fragments amplified by the three sets of RPA primers are all in line with the expected size.

[0051] 2.2 Evaluation of the effect of different RPA primers through CRISPR / Cas12a reaction The target gene fragments amplified by three sets of RPA primers were used as templates for CRISPR / Cas12a reactions. The CRISPR / Cas12a reaction system was as follows: 100nM LbCas12a, 2μL 10×NEBuffer 2.1, 400nMcrRNA1, 1000nM FAM-N7-BHQ1 ssDNA probe, 3μL RPA product, and pure water was added to make the final volume 20μL. Among them, the sequence of the reporter molecule FAM-N7-BHQ1 used was: 5'-FAM / TTTATTT / 3'-BHQ1.

[0052] After reacting at 37°C for 30 minutes, use a gel imager to visualize the reaction results under ultraviolet light. If the reaction solution shows green or light green fluorescence, the result is judged as positive; if the reaction solution is colorless, it is judged as negative. Results ( Figure 2 (B) shows the results of RPA-CRISPR / Cas12a using different RPA primers: the three sets of RPA primers can produce green fluorescence of the same intensity under ultraviolet light (shown as green in the original figure, because the attached figure cannot have color, Figure 2 B). Therefore, RPA1 primers (i.e., RPA1-F and RPA1-R in Table 2) were randomly selected for subsequent experiments. In the following, unless otherwise specified, the RPA primers used were RPA1-F and RPA1-R.

[0053] 2.3 Detection sensitivity Different copy numbers of gyrB-pUC19 plasmid (2.34×10 0 to 2.34×10 6 The target gene fragment was amplified by PCR, and the RPA amplification and CRISPR / Cas12a reaction were performed in sequence using the methods in “2.1 Amplifying the target gene fragment” and “2.2 Evaluating the effect of different RPA primers by CRISPR / Cas12a reaction”. The RPA primers used were RPA1-F and RPA1-R.

[0054] result( Figure 2 C shows the sensitivity test results of RPA-CRISPR / Cas12a observed under ultraviolet light using a gel imager) showing that the added copy number is 2.34×10 2 to 2.34×10 6 After the plasmid template was added, the reaction system could emit green fluorescence under ultraviolet light (shown as green in the original figure, because the attached figure cannot have color, Figure 2 C), indicating that the detection limit (LOD) of the RPA-CRISPR / Cas12a method using the system of the present invention is 2.34×10 2 copies / reaction. This detection limit is consistent with the fluorescence intensity value of fluorescence quantitative PCR detection ( Figure 2 D shows that the fluorescence changes were monitored by the real-time fluorescence quantitative PCR system, and the RPA-CRISPR / Cas12a sensitivity detection results obtained were consistent.

[0055] 2.4 Detection specificity The genomic DNA of Pseudomonas killing sweetfish, other Pseudomonas and common pathogens of marine fish were extracted as templates for RPA amplification. The methods in “2.1 Amplification of target gene fragments” and “2.2 Evaluation of the effects of different RPA primers by CRISPR / Cas12a reaction” were used to perform RPA amplification and CRISPR / Cas12a reaction in sequence. The RPA primers used were RPA1-F and RPA1-R.

[0056] result( Figure 2 E shows the specific detection results of RPA-CRISPR / Cas12a) shows that under ultraviolet light, only Pseudomonas plecoglossicida stimulated green fluorescence signals (shown as green in the original figure, because the attached figure cannot have color, Figure 2E), while Pseudomonas aeruginosa (P.aeruginosa), Pseudomonas putida (P.putida), Pseudomonas fluorescens (P.fluorescens), Vibrio parahaemolyticus (V.parahaemolyticus), Edwardsiella tarda (E.tarda), Vibrio alginolyticus (V.alginolyticus), Vibrio harveyi (V.harveyi), Streptococcus iniae (S.iniae) and Nocardia seriolae (N.seriolae) did not excite fluorescence, which indicates that the detection system of the present invention is highly specific to Pseudomonas killing sweetfish and can effectively avoid interference from various other Pseudomonas and common pathogens of marine fish.

[0057] Example 3: Detection of Pseudomonas aeruginosa by RPA-CRISPR / Cas12a-LFS In order to detect Pseudomonas aeruginosa in a portable and intuitive manner in practical applications, a visual detection method, namely RPA-CRISPR / Cas12a-LFS, was developed by combining the RPA-CRISPR / Cas12a reaction with a lateral flow chromatography test strip (LFS). The detection process is as follows Figure 3 As shown in A: The gyrB gene is amplified by RPA reaction, CRISPR / Cas12a reaction is performed, and the reaction solution is added to the lateral flow chromatography test strip (LFS). When the gyrB gene is present, after the FAM-N15-Biotin ssDNA probe is cut by Cas12a, FAM will bind to AuNPs-Anti-FAM, and then to the secondary antibody Anti-Mouse. Only the test line will show red, which is judged as positive; if the probe is not cut by Cas12a, Biotin will bind to streptavidin SA, and only the quality control line will show red, which is judged as negative; if only part of the probe is cut, then both the quality control line and the test line will show red, which is also judged as positive.

[0058] The sensitivity and specificity of the RPA-CRISPR / Cas12a-LFS-based detection method were evaluated as follows: 3.1 Detection sensitivity Different copy numbers of gyrB-pUC19 plasmid (2.34×10 0 to 2.34×10 6The RPA amplification and CRISPR / Cas12a reaction were performed in sequence using the methods in "2.1 Amplification of target gene fragments" and "2.2 Evaluation of the effects of different RPA primers by CRISPR / Cas12a reaction". The RPA primers used were RPA1-F and RPA1-R, and the reporter molecule FAM-N7-BHQ1 was replaced with FAM-N15-Biotin, with the sequence: 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin. After completing the CRISPR / Cas12a reaction, the obtained reaction solution was added dropwise to the lateral flow chromatography test strip for LFS detection.

[0059] result( Figure 3 B shows the sensitivity test results of RPA-CRISPR / Cas12a-LFS) shows that: using the system of the present invention, the detection limit based on the RPA-CRISPR / Cas12a-LFS method is 2.34×10 2 Copy / React.

[0060] 3.2 Detection specificity The genomic DNA of Pseudomonas aeruginosa and other common pathogens of marine fish was extracted as the template for RPA amplification. The methods in "2.1 Amplification of target gene fragments" and "2.2 Evaluation of the effects of different RPA primers by CRISPR / Cas12a reaction" were used to perform RPA amplification and CRISPR / Cas12a reaction in sequence. The RPA primers used were RPA1-F and RPA1-R, and the reporter molecule FAM-N7-BHQ1 was replaced with FAM-N15-Biotin, with the sequence: 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin. After completing the CRISPR / Cas12a reaction, the obtained reaction solution was added dropwise to the lateral flow chromatography test strip for LFS detection.

[0061] result( Figure 3 C shows the specific detection results of RPA-CRISPR / Cas12a-LFS) showing that only the LFS detection line containing Pseudomonas aeruginosa showed obvious bands, while there were no bands on the detection lines of other pathogens, indicating that the detection system of the present invention has high detection specificity for Pseudomonas aeruginosa.

[0062] Example 4: Detection of spiked samples and clinical samples using the RPA-CRISPR / Cas12a-LFS method 4.1 Testing of spiked samples Spleen tissues were obtained from 2 large yellow croakers that were not infected with Pseudomonas aeruginosa and 1×10 4CFU of Pseudomonas australis was isolated, and then genomic DNA was extracted from the sample as a template for RPA amplification. The methods in "2.1 Amplification of target gene fragments" and "2.2 Evaluation of the effects of different RPA primers by CRISPR / Cas12a reaction" were used to perform RPA amplification and CRISPR / Cas12a reaction in sequence. The RPA primers used were RPA1-F and RPA1-R, and the reporter molecule was FAM-N7-BHQ1, with the sequence: 5'-FAM / TTTATTT / 3'-BHQ1. The fluorescence intensity was monitored using a real-time fluorescence quantitative PCR system, and after the CRISPR / Cas12a reaction was completed, the reaction results were visualized under UV light using a gel imager. Next, the reporter molecule FAM-N7-BHQ1 was replaced with FAM-N15-Biotin, with the sequence: 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin, and the CRISPR / Cas12a reaction was performed according to the above method. The reaction solution obtained after the CRISPR / Cas12a reaction was added dropwise to the lateral flow chromatography test strip for LFS detection.

[0063] The results showed that only the spiked samples showed fluorescence signals ( Figure 4 A shows the test results of spiked samples obtained using a gel imager. Figure 4 B shows the results of spiked sample detection obtained using a real-time fluorescence quantitative PCR system), which is consistent with the results of LFS detection ( Figure 4 C shows the spiked sample test results obtained using LFS).

[0064] 4.2 Clinical sample testing Genomic DNA was extracted from the spleen of 12 clinical marine fish samples and used as a template for RPA amplification. The methods in "2.1 Amplification of target gene fragments" and "2.2 Evaluation of the effects of different RPA primers by CRISPR / Cas12a reaction" were used to perform RPA amplification and CRISPR / Cas12a reaction in sequence. The RPA primers used were RPA1-F and RPA1-R, and the reporter molecule was FAM-N7-BHQ1 with the sequence: 5'-FAM / TTTATTT / 3'-BHQ1. The fluorescence intensity was monitored using a real-time fluorescence quantitative PCR system. After the CRISPR / Cas12a reaction was completed, the reaction results were visualized under UV light using a gel imager. Next, the reporter molecule FAM-N7-BHQ1 was replaced with FAM-N15-Biotin, the sequence was: 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin, and the CRISPR / Cas12a reaction was performed according to the above method. The reaction solution obtained after the CRISPR / Cas12a reaction was added dropwise to the lateral flow chromatography test strip for LFS detection. The results showed that 7 clinical samples showed fluorescent signals ( Figure 4 D shows the clinical sample test results obtained using a gel imager. Figure 4 E shows the clinical sample test results obtained using the real-time fluorescence quantitative PCR system. Figure 4 F shows the clinical sample test results obtained using LFS).

[0065] The above test results of spiked samples and clinical samples show that the detection system of the present invention can be used to conveniently and quickly detect Pseudomonas aeruginosa in marine fish based on the RPA-CRISPR / Cas12a-LFS method.

[0066] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A composition for detecting Pseudomonas australis based on CRISPR / Cas12a, characterized in that: It comprises a primer set, crRNA and Cas12a protein; the crRNA comprises the sequence shown in SEQ ID NO.

3.

2. The composition according to claim 1, characterized in that The primer set is a PCR primer set, including: an upstream primer shown in SEQ ID NO.1 and a downstream primer shown in SEQ ID NO.

2.

3. The composition according to claim 1, characterized in that The primer set is an RPA primer set, comprising one of the following a) to c): a) an upstream primer shown in SEQ ID NO.6 and a downstream primer shown in SEQ ID NO.7; b) an upstream primer shown in SEQ ID NO.8 and a downstream primer shown in SEQ ID NO.9; c) an upstream primer shown in SEQ ID NO.10 and a downstream primer shown in SEQ ID NO.

11.

4. The composition according to claim 1, characterized in that Also included are lateral flow chromatography test strips.

5. The composition according to claim 1 or 4, characterized in that It also includes a reporter molecule; the sequence of the reporter molecule is: 5'-FAM / TTTATTT / 3'-BHQ1 or 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin.

6. A method for detecting Pseudomonas acutus for non-diagnostic purposes using the composition of any one of claims 1 to 5, characterized in that: The following steps are involved: 1) preparing a nucleic acid amplification system comprising the primer set, and performing nucleic acid amplification using the DNA extracted from the sample to be tested as a template to obtain an amplified product; 2) preparing a CRISPR / Cas12a reaction system comprising the crRNA and Cas12a protein, performing a CRISPR / Cas12a reaction using the amplified product as a template, and determining whether Pseudomonas australis is present in the sample to be tested based on the product.

7. The method according to claim 6, characterized in that In step 2), the CRISPR / Cas12a reaction system also includes a reporter molecule with a sequence of 5'-FAM / TTTATTT / 3'-BHQ1; the method for judging whether Pseudomonas aeruginosa is present in the sample to be tested based on the product is as follows: observing the product under ultraviolet light, the product showing green or light green fluorescence indicates that Pseudomonas aeruginosa is present in the sample to be tested, and the product showing colorless fluorescence indicates that Pseudomonas aeruginosa is not present in the sample to be tested.

8. The method according to claim 6, characterized in that In step 2), the CRISPR / Cas12a reaction system also includes a reporter molecule with a sequence of 5'-FAM / TTTTTTTATTTTTTT / 3'-Biotin; the method for judging whether Pseudomonas aeruginosa is present in the sample to be tested based on the product is as follows: the product is added to a lateral flow chromatography test strip, and the detection line shows red, indicating that Pseudomonas aeruginosa is present in the sample to be tested, and only the quality control line shows red, indicating that Pseudomonas aeruginosa is not present in the sample to be tested.

9. The method according to claim 6, characterized in that In step 1), the nucleic acid amplification system is a PCR amplification system or an RPA amplification system.

10. The method according to claim 6, characterized in that In step 2), the CRISPR / Cas12a reaction system also includes NEB buffer.

Images